Evaluación de partículas de copolímeros de ácido láctico y glicólico (plga) como adyuvantes para la inducción de una respuesta inmune celular citotóxica

Résumé

The design of effective vaccines against cancer or diseases caused by intracellular microorganisms require the generation of cellular immunity, characterized by an adequate differentiation of CD4+ and CD8+ cells into Th1 and cytotoxic T lymphocytes (CTL). The strategies used nowadays, based on the administration of recombinant antigens (Ag), are poorly immunogenic and require appropriate immunological ¿adjuvant¿ to intensify the immune response. The term ¿adjuvant¿ describes any substance, combination of substances or strategies that augmented the specific immunity to an Ag as compared to that induced by the Ag or the vaccine alone. The overall goal of this work was the optimization of the parameters for subcutaneous vaccination with PLGA particles to elicit Ag-specific CTL responses. We investigate the effect of particles size and CpG co-delivery. The Ag (ovalbumin) was encapsulated into 1 µm- microparticles (MP) or 200 nm nanoparticles (NP). CpG containing oligonucleotide was adsorbed or encapsulated in Ag loaded PLGA particles. The particles were prepared using TROMS, a modification of the multiple emulsion procedure, developed in our research team. OVA and CpG motifs were encapsulated with high efficiency without signs of degradation. The Ag specific CD8+lymphocyte stimulation was evaluated after one shot or two shots, both at local and systemic level, employing different immunological techniques: OVA-tetramers quantification, intracellular and extracellular IFN-ã production and in vivo cytolytic activity. Furthermore, we compared the efficacy of the different formulations to cure E.G7 established tumors in mice. The results showed that that administration of CpG sequences with encapsulated Ag resulted indispensable to generate CTL responses after a single vaccination. Without CpG, Ag-loaded PLGA particles need boosting to induce CD8+activation. However, the systemic immune response resulted to be stronger with CpG cooperation. CpG location in the particle highly affected their adjuvanticity. Therefore, particles encapsulating the Ag with CpG sequences adsorbed onto the surface were more effective than those co-encapsulating both, the Ag and CpG motifs. A functional dissociation was observed between local (evaluated in lymph node) and systemic (evaluated in spleen) immune response. Whether the systemic immune response was independent of the size of the particles, in the lymph nodes upon boosting the strongest CTL activation was achieved after administration of NP, with rather influence of CpG co-delivery. Finally, among the formulations evaluated, Ag-loaded NP with adsorbed CpG showed the highest effectiveness to cure E.G7 tumor-bearing mice. These results are discussed in terms of the influence of size and CpG in the three signals required for the optimal CD8+ stimulation (Ag uptake and cross-presentation (signal 1), DC maturation (signal 2) and IL-12 production (signal 3)). 96 h after sc administration Ag-positive DC were only detected in mice that received NP (signal 1). On the other hand, the highest IL-12 plasma levels were determined in mice injected with adsorbed CpG (signal 3). Other cells of innate immunity, mobilized after PLGA particles and CpG administration (i.e. NK cells) could complete this picture and explain the overall of the present work.